Resting Cell Neutralization Assay for HIV-1 Primary Isolates

A technique is described for detecting the activity of neutralizing polyclonal or monoclonal antibodies against HIV-1 primary isolates. Most commonly, neutralizing antibody activity for HIV-1 is assessed by quantifying the ability of antibodies to inhibit virus infection in mitogen-activated peripheral blood mononuclear cells or transformed lymphocytes. Because the target of HIV infectionin vivois neither a mitogen-activated nor a transformed cell, an assay using unstimulated peripheral blood mononuclear cells as a more physiologic target cell was developed. This “resting cell assay” mainly utilizes primary HIV-1 isolates that have been carried for only a few passagesin vitro.The result is an assay that is more efficient to perform and that detects neutralizing activity with comparable or greater sensitivity than that previously described for assays of primary HIV-1 isolates.     

A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID₅₀ ml⁻¹~10⁻⁴ EID₅₀ ml⁻¹,which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 μl of AIV H5N1 with 10⁻⁴ EID₅₀ ml⁻¹.To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT–PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT–PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal–tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.     

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Over the past 10 years, a growing field of research supporting the value of myeloperoxidase (MPO) as a prognostic indicator in acute cardiac pathophysiologies has emerged. The availability of a rapid and disposable MPO detection platform would enable research clinicians to more readily assess MPO indications for guiding therapy and also facilitate clinicians at the patient interface to readily adopt MPO testing and potentially drive more informed prognoses. Here we describe the isolation of a high-affinity avian MPO-specific recombinant antibody panel using phage display. Rapid isolation of a suitable single-chain variable fragment (scFv) antibody was facilitated using a surface plasmon resonance (SPR)-based “off-rate ranking” screening process. The selected scFv was then successfully incorporated into a rapid, simple, and sensitive one-step lateral flow immunoassay (LFIA) for the detection of MPO. This “one-step” feature of the developed assay was made possible by the scFv’s strong affinity for MPO, obviating the need for sandwich signal enhancement steps. The assay’s rapid performance was also further enhanced by exploiting the intrinsic enzymatic properties of MPO in its final detection. Use of the optimized LFIA facilitated the sensitive detection of MPO in MPO-depleted serum within clinically relevant reference ranges.     

Crystal violet as an i-motif structure probe for reversible and label-free pH-driven electrochemical switch

A simple pH-induced electrochemical switch based on an i-motif structure is developed by using crystal violet as a selective electrochemical probe for the i-motif structure. Thiol-modified cytosine-rich single-strand oligonucleotide (C-rich ssDNA) can be self-assembled on the gold electrode surface via gold–sulfur interaction. Crystal violet is employed as an electrochemical probe for the i-motif structure because of its capability of binding with the i-motif structure through an end-stacking mode. In acidic aqueous solution, crystal violet may approach the electrode surface owing to the formation of the i-motif structure, resulting in an obvious signal, so-called “ON” state. Whereas in neutral or basic aqueous solution, the i-motif structure unfolds to dissociative single strand, which causes crystal violet to leave from the electrode surface, and a weak signal is obtained, so-called “OFF” state. In addition, in the range of pH 4.6–7.3, the increase in current has a good linear relationship (R = 0.989) with pH value in the testing solutions. This pH-driven electrochemical switch has the advantages of simplicity, sensitivity, high selectivity, and good reversibility. Furthermore, it provides a possible platform for pH measurement.     

A modified protein assay from microgram to low nanogram levels in dilute samples

In this article, we present a modified and improved protein assay that was previously described as “amidoschwarz assay” by Schaffner and Weissmann [13]. Our improved protein assay is user-friendly and 30–40 times more sensitive than the earlier method. The assay was developed into three formats (macro-, micro-, and nanoassay) with trichloroacetic acid (TCA) as protein precipitating agent, measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots bound to nitrocellulose membrane with lowest protein measurements to 1 and 0.1 μg, respectively. On the other hand, the nanoassay, with combination staining of amido black followed by colloidal gold, can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 μg/ml prior to their biochemical analysis such as in comparative proteomics.     

Detection of rViscumin in plasma samples by immuno-PCR

To allow for pharmacokinetic studies in adjunction with the current clinical developments of the potent cytostatic anti-cancer drug rViscumin, a sandwich immuno-PCR (IPCR) assay was developed for the detection of rViscumin in blood plasma. The IPCR was carried out with a commercially available reagent kit, consisting of pre-assembled rViscumin-specific antibody-DNA conjugates as well as a specific competitor DNA fragment to be amplified by PCR. Various combinations of capture- and detection-antibodies were compared for performance in IPCR. Using the optimized assay, as few as 50 zeptomol (approx. 100 fg/ml) rViscumin (MW 57 kDa) was detectable in standardized human serum samples. The IPCR assay was very selective for rViscumin and in spiking experiments in proband plasma samples, signal recovery rates between 70% and 120% were obtained. The linear sensitivity range of the assay covered more than five orders of magnitude. Repeated measurements of rViscumin resulted in a mean standard deviation value of 14.2%.     

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml^?1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.     

Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)–V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K_D = 0.93 nM to K_D = 348 nM). That decrease in the affinity of drug–ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug–ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.     

报道基因SEAP的应用

分泌型碱性磷酸酶(secreted alkaline phosphatase,SEAP)由于既有很高的灵敏性,又可分泌至培养介质中,因而SEAP基因成为一种广泛使用的报道基因。目前,该基因在中和抗体检测试验、实时监测分析及工程抗体构建与检测方法有较多应用。该基因在双报道基因系统中运用,可同时起到定性定量作用。本文将对报道基因SEAP的使用原理与应用加以综述。     

Graphene oxide based fluorescent aptasensor for adenosine deaminase detection using adenosine as the substrate

We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong π-π stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development.     

Baroreflex variability and “resetting”: A new perspective

A new framework is proposed for the interpretation of spontaneous cardiac baroreflex sensitivity data and the general concept of baroreflex resetting. The framework is used to explore baroreflex function along two separate lines of inquiry: one following a direct intervention in baroreflex function in individual subjects, another in a group of subjects where baroreflex function may have been compromised by coronary artery disease or aging. It is found that under baseline conditions the baroreflex is in a “free-floating” state in which the gain or “sensitivity” is highly variable, while under orthostatic stress or in the absence of or reduced vagal input the gain is more tightly controlled with an expected decline in sensitivity but a very large decline in the variability of that sensitivity. It is concluded that baroreflex “resetting” is better viewed not simply as a change in baroreflex sensitivity but rather as a change in the “focus” or “attention” of the baroreflex as expressed by an observed decline in the variability of the measured gain. The results do not support the interpretation of baroreflex “resetting” as a departure from or return to a universal “set point” as in homeostasis or open loop models.     

Targeting Notch signaling pathway to overcome drug resistance for cancer therapy

Chemotherapy is an important therapeutic strategy for cancer treatment and remains the mainstay for the management of human malignancies; however, chemotherapy fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Recently, emerging evidences suggest that Notch signaling pathway is one of the most important signaling pathways in drug-resistant tumor cells. Moreover, down-regulation of Notch pathway could induce drug sensitivity, leading to increased inhibition of cancer cell growth, invasion, and metastasis. This article will provide a brief overview of the published evidences in support of the roles of Notch in drug resistance and will further summarize how targeting Notch by “natural agents” could become a novel and safer approach for the improvement of tumor treatment by overcoming drug resistance.     

Configural processing of different topologically structured figures: an ERP Study

According to Chen’s theory, topological differences are perceived faster than feature differences in early visual perception. We hypothesized that topological perception is caused by the sensitivity in discriminating figures with and without “holes”. An ERP experiment was conducted utilizing a passive paradigm to investigate the differences in perceiving figures with “hole” and with “no-hole”. The results showed differences in N170 components between figures with “holes” and with “no-holes”. The inversion of the “hole” could influence the latency of N170, but the inversion of the “no-hole” could not, which indicated that global features are processed first in the “hole” perception whilst local features are given priority to the “no-hole” perception. This result was similar to studies concerning face and non-face objects, suggesting a configural processing of the “hole”.     

A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.     

Fluorescence intercalator displacement assay for screening G4 ligands towards a variety of G-quadruplex structures

The potential formation of G-quadruplexes in many regions of the genome makes them an attractive target for drug design. A large number of small molecules synthesized in recent years display an ability to selectively target and stabilize G-quadruplexes. To screen for G4 ligands, we modified a G4-FID (G-quadruplex Fluorescent Intercalator Displacement) assay. This test is based on the displacement of an “on/off” fluorescence probe, Thiazole Orange (TO), from quadruplex or duplex DNA matrices by increasing amounts of a putative ligand. Selectivity measurements can easily be achieved by comparing the ability of the ligand to displace TO from various quadruplex and duplex structures. G4-FID requires neither modified oligonucleotides nor specific equipment and is an isothermal experiment. This test was adapted for high throughput screening onto 96-well plates allowing the comparison of more than twenty different structures. Fifteen different known G4 ligands belonging to different families were tested. Most compounds showed a good G4 vs duplex selectivity but exhibited little, if any, specificity for one quadruplex sequence over the others. The quest for the “perfect” specific G4 ligand is not over yet!     

Non-radioactive detection of β-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC

Summary The use of transient gene expression assays for the study of natural or engineered plant promoters is affected by a considerable degree of inter-experiment variability. As a means of obtaining interpretable data from a limited number of experiments, we worked out conditions for the simultaneous determi nation of the activity of two reporter genes, a sample and a reference, ona single extract of co-transformed protoplasts. ß-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) genes, both under the control of the CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on two independent plasmids. The parallel expression of the two reporter genes in several independent co-transformation experiments was verified. Conditions for the use of a single protoplast extraction buffer and for the simultaneous assay of both reporter gene activities were set up. A HPLC method for the non-radioactive determination of both enzyme activities on a single aliquot of the reaction mixture was developed. The resulting procedure was tested using the GUS gene as reference and the CAT gene, under the control of either wild type or upstream-deleted (–90) CaMV 35S promoter, as sample. The protocol is simple and allows the fast analysis of plant promoters in the presence of a true internal standard under conditions in which assay manipulations are reduced to a minimum and both reporter gene activities are subjected to the same experimental treatments.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyl transferase - EDTA ethylenediaminetetraacetic acid - GUS ß-glucuronidase - HPLC high performance liquid chromatography - MES 2-morpholinoethanesulphonic acid - MS medium after Murashige and Skoog (1962) - MUG 4-methyl umbelliferyl glucuronide - MU methylumbelliferone - NOS nopaline synthase - PEG polyethylene glycol - TRIS tris-hydroxymethyl aminomethane - UV ultraviolet